Process and reagent for determining the activity of chymotrypsin and trypsin in feces

Abstract

The present invention provides a process for the determination of the activity of chymotrypsin or trypsin in feces by measurement of the rate of fission of an appropriate substrate by a fecal suspension in an aqueous or aqueous-organic medium, wherein a fecal sample is suspended in the presence of a surface-active agent. The present invention also provides a reagent for carrying out this process, comprising a surface-active agent, an enzyme substrate and an aqueous salt solution, said reagent having a pH value of from 7 to 11.

What is claimed is: 
     
       1. A process for the determination of chymotrypsin or trypsin in a feces sample, comprising the steps of: suspending the feces sample in an aqueous or aqueous-organic suspension medium including one or more water soluble salts in an amount whereby the ionic strength of the salts from 20 to 1000 mval/liter, and from 0.02% to 10% by weight of a surface-active agent selected from the group consisting of cationic and non-ionic tensides;   contacting the suspended sample with a substrate specific for the determination of chymotrypsin or trypsin by measurement of the rate of fission of the substrate; and   photometrically measuring the rate of fission of the substrate as a measure of the activity of chymotrypsin or trypsin in the suspension.   
     
     
       2. The process of claim 1 wherein the surface active agent is a quaternary ammonium salt or an alkyl pyridinium salt. 
     
     
       3. The process of claim 2, wherein the quaternary ammonium salt is lauryl trimethyl ammonium chloride. 
     
     
       4. The process of claims 1 or 2, wherein the concentration of the surface-active agent is 0.5 to 5% by weight. 
     
     
       5. The process of claim 1, wherein the surface-active agent is a non-ionic tenside selected from the group consisting of a polyether, a fatty acid amide, a fatty amine, a fatty alcohol, a polymer of propylene and ethylene oxide, a polyoxyethylene sorbitan monooleate or laurate, an addition product of propylene oxide/ethylenediamine/ethylene oxide, an amide oxide or fatty acid ester of polyalcohols and tallow alcohol polyglycol ethers, or   a cationic tenside selected from the group consisting of a straight-chained or cyclic ammonium compound, a benzalkonium chloride or other quaternary ammonium salt, an amine salt, a pyridinium salt and a quaternary fatty amine polyglycol ether.   
     
     
       6. The process of claim 1, wherein the fission of the substrate forms amino acids, further comprising determining the amino acid formed by the fission. 
     
     
       7. The process of claim 6, wherein the amino acid is determined by titration with a base. 
     
     
       8. The process of claim 1, wherein the water-soluble salt is a sodium salt, a calcium salt, or a mixture thereof. 
     
     
       9. The process of claim 1, wherein the suspension medium comprises 0.02 to 10% by weight of a surface-active agent, 20 to 1000 mval/liter of a water-soluble salt, 10 to 1000 mmol/liter of a buffer (pH 7 to 11) and 0.05 and to 5 mmol/liter enzyme substrate is used. 
     
     
       10. The process of claim 1, wherein the suspension medium comprises 0.5 to 5% by weight of surface-active agent; 100 to 1100 mmole/liter of sodium chloride, 20 to 500 mmole/liter of calcium chloride, or a mixture of 100 to 1100 mmole/liter of sodium chloride and 20 to 500 mmole/liter of calcium chloride; and 50 to 200 mmole/liter of tris buffer (pH 8 to 10); and 0.2 to 2 mmoles/liter of enzyme substrate are used. 
     
     
       11. The process of claim 1, 31 or 32, wherein chymotrypsin is to be determined. 
     
     
       12. The process of claim 11, wherein the substrate used is Ala-Ala-Phe-pNA, Succ-Ala-Ala-Pro-Phe-pNa or MeO-Succ-Arg-Pro-Tyr-pNA. 
     
     
       13. The process of claim 1, 31 or 32, wherein trypsin is determined. 
     
     
       14. The process of claim 13, wherein the substrate used is carbobenzoxy-Val-Gly-Arg-pNA acetate. 
     
     
       15. The process of claim 1, further comprising centrifuging the suspended sample to remove suspended particles therefrom, the determining of chymotrypsin or trypsin being accomplished on a predetermined portion of the supernatant aqueous medium resulting from the centrifugation.